1. Field of the Invention
The present invention relates to identification of filamentous microorganisms, particularly to the identification of those filamentous microorganisms which cause sludge bulking during waste water treatment.
2. Description of the Related Art
Most waste water processing stations use activated sludge technology as their main bio-process. However, due to the nature of industrial waste water, the bio-process itself has some inherent problems, particularly with excess foaming and sludge bulking Sludge bulking is mainly caused by the uncontrolled growth of filamentous microorganisms which result in the structural destruction of the flocs, and in their decrease subsidence. Meanwhile, because identifying filamentous microorganisms that cause sludge bulking is a timely process, filamentous microorganisms are typically not well controlled in waste water treatment resulting in poorer water quality.
Note that by expediently identifying filamentous microorganisms which cause sludge bulking in waste water treatment, prophylaxis can be enhanced. However, in conventional activated sludge systems, the bacteria flora is very complicated and classification of bacteria causing bulking cannot be made by morphology or growth condition. Therefore, conventional identification methods have their shortcomings.
There are mainly two kinds of high throughput identification methods for microorganisms. The first one is immunoassay. Immunoassay utilizes specific antibodies to a microorganism for the identification of that particular microorganism. The second one is through detection of a target gene of the particular microorganism of interest. For example, a specific gene of certain microorganism can be detected using polymerase chain reaction (PCR) or DNA hybridization (Southern blotting). However, PCR can identify only one or a few microorganisms at a time, and multiple primers are needed to identify different microorganisms. For identification of the filamentous microorganisms which causes sludge bulking in waste water treatment, multiplex PCR or serial PCRs is required. However, multiplex PCR has some disadvantages, such as decreased PCR sensitivity (Chang et al., 2001). In addition, performing serial PCRs increases assay complexity, manpower and cost. Therefore, the application of multiplex PCR for the identification of filamentous microorganisms in waste water treatment has been limited.
Bacteria contain both highly conserved and highly variable regions of DNA. Depending on the bacteria of interest and on the region of interest, the DNA for that region will either be highly variable or highly conserved. The length of the 16S rRNA gene (16S rDNA) of all bacteria is about 1.5 Kb and typically conserved. In comparison with the 16S rDNA, the length of the 23S rRNA is about 3.0 Kb and typically more variable. Different bacteria vary to a large degree in both length and sequence of the 16S-23S ribosomal DNA intergenic spacer (ITS) region. The length of the 16S-23 S ribosomal intergenic spacer region ranged from 60 to 1529 bp, and it was proposed by Gürtler that the intergenic spacer region can be used as an identification tool of microorganisms (Gürtler and Stanisich, 1996). Except for a few bacteria, the intraspecies sequence similarities of the 16S-23S ribosomal intergenic spacer are high, but interspecies sequence similarities are low. In comparison with the 16S rDNA sequence, wherein the sequencing of the 16S rDNA usually needs to be performed twice, the 16S-23S ribosomal DNA intergenic spacer region sequence is shorter and only requires one sequencing of the 16S-23S ribosomal DNA intergenic spacer region sequence to obtain the entire sequence. The divergence between interspecies of the 16S-23S ribosomal DNA intergenic spacer region sequence is greater than that of the 16 rDNA, (the similarities of the 16 rDNA between some bacteria can be as much as and even greater than 99%). Accordingly, Kawamura and Patel considered that the 16S-23S ribosomal DNA intergenic spacer region sequence is helpful for microorganism identification (Kawamura et al., 1995; Patel et al., 1998).
The present invention relates to a new method for identifying filamentous microorganisms which causes sludge bulking in waste water treatment via use of the 16S-23S ribosomal DNA intergenic spacer sequence. The new method involves amplifying the DNA of target microorganisms using PCR, and then detecting the PCR product by oligonucleotide sequence from the 16S-23S ribosomal DNA intergenic spacer sequence which are used as probes on microarrays. Currently, only Kim (Kim et al., 2004) uses a DNA chip having oligonucleotide sequences from the 16S rDNA sequence to detect filamentous microorganisms in activated sludge. Meanwhile, the intergenic spacer region sequence (ITS) has been mainly used to identify bacteria for some pathogens in biology and medicine, such as Entercoccus species (Tung et al., 2006), E. coli (Lin et al., 2005), Granulicatella species (Tung et al., 2006), Pseudomonas aeruginosa (Lin et al., 2005), Streptococcus species (Tung et al., 2006), etc. The intergenic spacer region sequence has not been applied for detecting filamentous microorganisms in activated sludge.